Impact of age at onset and newborn screening on outcome in organic acidurias.

BACKGROUND AND AIM: To describe current diagnostic and therapeutic strategies in organic acidurias (OADs) and to evaluate their impact on the disease course allowing harmonisation. METHODS: Datasets of 567 OAD patients from the E-IMD registry were analysed. The sample includes patients with methylmalonic (MMA, n = 164), propionic (PA, n = 144) and isovaleric aciduria (IVA, n = 83), and glutaric aciduria type 1 (GA1, n = 176). Statistical analysis included description and recursive partitioning of diagnostic and therapeutic strategies, and odds ratios (OR) for health outcome parameters. For some analyses, symptomatic patients were divided into those presenting with first symptoms during (i.e. early onset, EO) or after the newborn period (i.e. late onset, LO). RESULTS: Patients identified by newborn screening (NBS) had a significantly lower median age of diagnosis (8 days) compared to the LO group (363 days, p < 0.001], but not compared to the EO group. Of all OAD patients 71 % remained asymptomatic until day 8. Patients with cobalamin-nonresponsive MMA (MMA-Cbl(-)) and GA1 identified by NBS were less likely to have movement disorders than those diagnosed by selective screening (MMA-Cbl(-): 10 % versus 39 %, p = 0.002; GA1: 26 % versus 73 %, p < 0.001). For other OADs, the clinical benefit of NBS was less clear. Reported age-adjusted intake of natural protein and calories was significantly higher in LO patients than in EO patients reflecting different disease severities. Variable drug combinations, ranging from 12 in MMA-Cbl(-) to two in isovaleric aciduria, were used for maintenance treatment. The effects of specific metabolic treatment strategies on the health outcomes remain unclear because of the strong influences of age at onset (EO versus LO), diagnostic mode (NBS versus selective screening), and the various treatment combinations used. CONCLUSIONS: NBS is an effective intervention to reduce time until diagnosis especially for LO patients and to prevent irreversible cerebral damage in GA1 and MMA-Cbl(-). Huge diversity of therapeutic interventions hampers our understanding of optimal treatment.

Glut1 deficiency syndrome and novel ketogenic diets.

The classical ketogenic diet has been used for refractory childhood epilepsy for decades. It is also the treatment of choice for disorders of brain energy metabolism, such as Glut1 deficiency syndrome. Novel ketogenic diets such as the modified Atkins diet and the low glycemic index treatment have significantly improved the therapeutic options for dietary treatment. Benefits of these novel diets are increased palatability, practicability, and thus compliance-at the expense of lower ketosis. As high ketones appear essential to meet the brain energy deficit caused by Glut1 deficiency syndrome, the use of novel ketogenic diets in this entity may be limited. This article discusses the current data on novel ketogenic diets and the implications on the use of these diets in regard to Glut1 deficiency syndrome.

Aminoacidopathies and organic acidurias in Tunisia: a retrospective survey over 23 years.

BACKGROUND: Inborn errors of metabolism are neglected in developing countries because they are not as common as infectious and nutritional disorders. In Tunisia, no information is available on the incidence and epidemiological features of these inherited metabolic diseases. AIMS: To precise the profile of aminoacidopathies other than phenylketonuria and organic acidurias and to estimate their incidences in Tunisia. METHODS: Between 1987 and 2009, our laboratory received 13171 requests for analysis of patients with symptoms suggestive of inborn errors of metabolism. For these cases, ion exchange chromatography of free amino acids was performed on amino acids analyser. Urinary organic acids profiles were determined by gas chromatography-mass spectrometry. RESULTS: Abnormal cases were 370 (2.8%), divided into 212 cases of aminoacidopathies (57.3%) and 158 cases of organic acidurias (42.7%). The most frequent aminoacidopathies, were maple syrup disease (32.5%), tyrosinemia type I (28.8%) and nonketotic hyperglycinemia (16%). Methylmalonic aciduria (33.5%), propionic aciduria (18.4%) and 2-hyrdoxy glutaric aciduria (10.8%) were the most frequent organic acidurias. The incidences were calculated using the Hardy-Weinberg formula and were estimated at 1/13716 for maple syrup disease, 1/14804 for tyrosinemia type I, 1/16144 for methylmalonic aciduria and 1/23176 for propionic aciduria. CONCLUSION: Aminoacidopathies and organic acidurias turned out to be highly frequent in Tunisia, mainly because of a high rate of consanguinity. We believe that they are underestimated. To improve their diagnosis, it is necessary to have available sophisticated equipment which would allow early treatment of patients.

Long-term follow-up and treatment in nine boys with X-linked creatine transporter defect.

The creatine transporter (CRTR) defect is a recently discovered cause of X-linked intellectual disability for which treatment options have been explored. Creatine monotherapy has not proved effective, and the effect of treatment with L-arginine is still controversial. Nine boys between 8 months and 10 years old with molecularly confirmed CRTR defect were followed with repeated (1)H-MRS and neuropsychological assessments during 4-6 years of combination treatment with creatine monohydrate, L-arginine, and glycine. Treatment did not lead to a significant increase in cerebral creatine content as observed with H(1)-MRS. After an initial improvement in locomotor and personal-social IQ subscales, no lasting clinical improvement was recorded. Additionally, we noticed an age-related decline in IQ subscales in boys affected with the CRTR defect.

Treatment by oral creatine, L-arginine and L-glycine in six severely affected patients with creatine transporter defect.

BACKGROUND: X-linked cerebral creatine deficiency is caused by the deficiency of the creatine transporter (CTP) encoded by the SLC6A8 gene. PATIENTS AND METHODS: We report here a series of six patients with severe CTP deficiency, four males and two females; clinical presentations include mild to severe mental retardation (6/6), associated with psychiatric symptoms (5/6: autistic behaviour, chronic hallucinatory psychosis), seizures (2/6) and muscular symptoms (2/4 males). Diagnosis was suspected upon elevated urinary creatine/creatinine (except in one of the female patients) and on a markedly decreased creatine peak on magnetic resonance spectroscopy (MRS). Diagnosis was confirmed by molecular analysis that identified four novel mutations not reported so far, including a mutation found twice in two male patients. All patients were treated successively and according to the same protocol by creatine alone then combined to its precursors, L-glycine and L-arginine for 42 months. RESULTS AND CONCLUSION: In our patients, creatine supplementation alone or with its precursors L-glycine and L-arginine showed benefit only in the muscular symptoms of the disease and no improvement in the cognitive and psychiatric manifestations and did not modify brain creatine content on MRS of male and female CTP deficient patients. New treatment strategies are required including creatine derivatives transported independently from CTP or using alternative pathways and transporters.

Creatine transporter deficiency in two adult patients with static encephalopathy.

Creatine transporter deficiency is a recently identified X-linked inborn error of metabolism. The natural course of the disease is not well delineated since clinical data from adult patients have scarcely been reported. A progressive course of the disease has been noted in a few described cases. We report the first two Spanish adult patients with creatine transporter deficiency and compare their clinical phenotype and the evolution of the disease with those of other published cases. The two brothers were identified in a study of a cohort of 610 mentally handicapped male patients. The disease was detected by biochemical studies and confirmed by DNA studies. The most significant clinical features were mental retardation, epilepsy and autistic behaviour, and these symptoms did not worsen, in contrast to other reports. They did not present gastrointestinal problems or movement disorders. Creatine transporter deficiency could be an underdiagnosed metabolic disorder and should be considered in adult patients with mental retardation. Clinical presentation of this disorder showed marked differences among adult patients and the course of the disease was static in our cases. Detection of additional adult patients might allow better understanding of the phenotypic outcome at a later age.

Citrin deficiency, a perplexing global disorder.

Citrin deficiency, caused by mutations in SLC25A13, can present with neonatal intrahepatic cholestasis or with adult onset neuropsychiatric, hepatic and pancreatic disease. Until recently, it had been thought to be found mostly in individuals of East Asian ancestry. A key diagnostic feature has been the deficient argininosuccinate synthetase (ASS) activity (E.C. 6.3.4.5) in liver, with normal activity in skin fibroblasts. In this series we describe the clinical presentation of 10 patients referred to our laboratories for sequence analysis of the SCL25A13 gene, including several patients who presented with elevated citrulline on newborn screening. In addition to sequence analysis performed on all patients, ASS enzyme activity, citrulline incorporation and Western blot analysis for ASS and citrin were performed on skin fibroblasts if available. We have found 5 unreported mutations including two apparent founder mutations in three unrelated French-Canadian patients. In marked contrast to previous cases, these patients have a markedly reduced ASS activity in skin fibroblasts. The presence of citrin protein on Western blot in three of our cases reduces the sensitivity of a screening test based on protein immunoblotting. The finding of citrin mutations in patients of Arabic, Pakistani, French Canadian and Northern European origins supports the concept that citrin deficiency is a panethnic disease.

Novel SLC7A7 large rearrangements in lysinuric protein intolerance patients involving the same AluY repeat.

Lysinuric protein intolerance (LPI) is a rare autosomal inherited disease caused by defective cationic aminoacid transport 4F2hc/y(+)LAT-1 at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is a multisystemic disease with a variety of clinical symptoms such as hepatosplenomegaly, osteoporosis, hypotonia, developmental delay, pulmonary insufficiency or end-stage renal disease. The SLC7A7 gene, which encodes the y(+)LAT-1 protein, is mutated in LPI patients. Mutation analysis of the promoter localized in intron 1 and all exons of the SLC7A7 gene was performed in 11 patients from 9 unrelated LPI families. Point mutation screening was performed by exon direct sequencing and a new multiplex ligation probe amplification (MLPA) assay was set up for large rearrangement analysis. Eleven SLC7A7-specific mutations were identified, seven of them were novel: p.L124P, p.C425R, p.R468X, p.Y274fsX21, c.625+1G>C, DelE4-E11 and DelE6-E11. The novel large deletions originated by the recombination of Alu repeats at introns 3 and 5, respectively, with the same AluY sequence localized at the SLC7A7 3' region. The novel MLPA assay is robust and valuable for LPI molecular diagnosis. Our results suggest that genomic rearrangements of SLC7A7 play a more important role in LPI than has been reported, increasing the detection rate from 5.1 to 21.4%. Moreover, the 3' region AluY repeat could be a recombination hot spot as it is involved in 38% of all SLC7A7 rearranged chromosomes described so far.

Iminoglycinuria and hyperglycinuria are discrete human phenotypes resulting from complex mutations in proline and glycine transporters.

Iminoglycinuria (IG) is an autosomal recessive abnormality of renal transport of glycine and the imino acids proline and hydroxyproline, but the specific genetic defect(s) have not been determined. Similarly, although the related disorder hyperglycinuria (HG) without iminoaciduria has been attributed to heterozygosity of a putative defective glycine, proline, and hydroxyproline transporter, confirming the underlying genetic defect(s) has been difficult. Here we applied a candidate gene sequencing approach in 7 families first identified through newborn IG screening programs. Both inheritance and functional studies identified the gene encoding the proton amino acid transporter SLC36A2 (PAT2) as the major gene responsible for IG in these families, and its inheritance was consistent with a classical semidominant pattern in which 2 inherited nonfunctional alleles conferred the IG phenotype, while 1 nonfunctional allele was sufficient to confer the HG phenotype. Mutations in SLC36A2 that retained residual transport activity resulted in the IG phenotype when combined with mutations in the gene encoding the imino acid transporter SLC6A20 (IMINO). Additional mutations were identified in the genes encoding the putative glycine transporter SLC6A18 (XT2) and the neutral amino acid transporter SLC6A19 (B0AT1) in families with either IG or HG, suggesting that mutations in the genes encoding these transporters may also contribute to these phenotypes. In summary, although recognized as apparently simple Mendelian disorders, IG and HG exhibit complex molecular explanations depending on a major gene and accompanying modifier genes.

Inherited epithelial transporter disorders--an overview.

In the late 1990s, the identification of transporters and transporter-associated genes progressed substantially due to the development of new cloning approaches such as expression cloning and, subsequently, to the implementation of the human genome project. Since then, the role of many transporter genes in human diseases has been elucidated. In this overview, we focus on inherited disorders of epithelial transporters. In particular, we review genetic defects of the genes encoding glucose transporters (SLC2 and SLC5 families) and amino acid transporters (SLC1, SLC3, SLC6 and SLC7 families).

Nephropathy advancing to end-stage renal disease: a novel complication of lysinuric protein intolerance.

OBJECTIVE: To analyze systemically the prevalence of renal involvement in a cohort of Finnish patients with lysinuric protein intolerance (LPI) and to describe the course and outcome of end-stage renal disease in 4 patients. STUDY DESIGN: The clinical information in a cohort of 39 Finnish patients with LPI was analyzed retrospectively. RESULTS: Proteinuria was observed in 74% of the patients and hematuria was observed in 38% of the patients during follow-up. Elevated blood pressure was diagnosed in 36% of the patients. Mean serum creatinine concentration increased in 38% of the patients, and cystatin C concentration increased in 59% of the patients. Four patients required dialysis, and severe anemia with poor response to erythropoietin and iron supplementation also developed in these patients. CONCLUSIONS: Our findings suggest that renal function of patients with LPI needs to be carefully monitored, and hypertension and hyperlipidemia should be treated effectively. Special attention also should be paid to the prevention of osteoporosis and carnitine deficiency in the patients with end-stage renal disease associated with LPI. The primary disease does not prohibit treatment by dialysis and renal transplantation.

Successful whole lung lavage in pulmonary alveolar proteinosis secondary to lysinuric protein intolerance: a case report.

BACKGROUND: Pulmonary alveolar proteinosis (PAP) is a rare disease characterised by accumulation of lipoproteinaceous material within alveoli, occurring in three clinically distinct forms: congenital, acquired and secondary. Among the latter, lysinuric protein intolerance (LPI) is a rare genetic disorder caused by defective transport of cationic amino acids. Whole Lung Lavage (WLL) is currently the gold standard therapy for severe cases of PAP. CASE PRESENTATION: We describe the case of an Italian boy affected by LPI who, by the age of 10, developed digital clubbing and, by the age of 16, a mild restrictive functional impairment associated with a high-resolution computed tomography (HRCT) pattern consistent with pulmonary alveolar proteinosis. After careful assessment, he underwent WLL. CONCLUSION: Two years after WLL, the patient has no clinical, radiological or functional evidence of pulmonary disease recurrence, thus suggesting that WLL may be helpful in the treatment of PAP secondary to LPI.

Overexpression of wild-type creatine transporter (SLC6A8) restores creatine uptake in primary SLC6A8-deficient fibroblasts.

In the study reported, we prove that mutations in the SLC6A8 gene are responsible for SLC6A8 deficiency, a cerebral creatine deficiency syndrome (CCDS), since overexpression of the wild-type SLC6A8 open reading frame (ORF) restores the creatine uptake profile in SLC6A8-deficient fibroblasts.

Hazards associated with pregnancies and deliveries in lysinuric protein intolerance.

Lysinuric protein intolerance (LPI) is an autosomal recessive transport disorder of the dibasic amino acids. The defect leads to deficiency of lysine, arginine, and ornithine and, secondarily, to a functional disorder of the urea cycle. Transient postprandial hyperammonemia and subsequent persistent protein aversion, linked with several other biochemical and clinical characteristics of the disease, suggest an increased risk for maternal and fetal complications during pregnancy and delivery. Our unique material on the outcomes of 18 pregnancies of 9 Finnish mothers with LPI and the follow-up of their 19 children shows that maternal LPI is truly associated with increased risk of anemia, toxemia, and intrauterine growth retardation during pregnancy and bleeding complications during delivery. Successful pregnancies and deliveries can still be achieved with careful follow-up of blood pressure and laboratory values. The children of the mothers with LPI generally develop normally. Special care of maternal protein nutrition and control of ammonemia, anemia, and toxemia during pregnancy are essential. We propose centralization of deliveries to obstetric units with capability to deal with bleeding complications and rare inborn errors of metabolism.

Lysinuric protein intolerance: identification and functional analysis of mutations of the SLC7A7 gene.

Lysinuric protein intolerance (LPI) is an inherited hyperdibasic aminoaciduria caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in the intestine and kidney. LPI is relatively common in Finland and a few clusters of patients are known in Italy and Japan. The SLC7A7 gene, mutated in LPI patients, encodes the y+LAT-1 protein which is the light subunit of a heterodimeric CAA transporter. We performed the mutation analysis in seven probands from five unrelated LPI families and identified five novel SLC7A7 mutations (p.M50K, p.T188I, p.R333M, p.Y457X, and c.499+?_629-?). By expression studies in X. laevis oocytes or patient's renal tubular cells, the functional analysis of altogether eight SLC7A7 mutations is here reported. Noteworthy, the p.R333M mutation, caused by a G to T transversion of the last nucleotide at 3' end of exon 7, disrupts a functional splicing motif generating misspliced transcripts. Three of the novel mutations were found in patients originating from Greece and Pakistan thus increasing the list of ethnic backgrounds where LPI mutant alleles are present. This reinforces the view that the rarity of LPI outside Finland might be ascribed to misdiagnosis of this disease.

Recurrent fatal pulmonary alveolar proteinosis after heart-lung transplantation in a child with lysinuric protein intolerance.

We present a case of recurrent pulmonary alveolar proteinosis after heart-lung transplantation in a child with lysinuric protein intolerance. The recurrence of the pulmonary disease provides further insight regarding the possible pathogenesis of pulmonary alveolar proteinosis and therapeutic options for this complication.

Expression of heteromeric amino acid transporters along the murine intestine.

Members of the new heterodimeric amino acid transporter family are composed of two subunits, a catalytic multitransmembrane spanning protein (light chain) and a type II glycoprotein (heavy chain). These transporters function as exchangers and thereby extend the transmembrane amino acid transport selectivity to specific amino acids. The heavy chain rBAT associates with the light chain b degrees (,+)AT to form a cystine and cationic amino acid transporter. The other heavy chain, 4F2hc, can interact with seven different light chains to form various transporters corresponding to systems L, y(+)L, asc or x(-)(c). The importance of some of these transporters in intestinal and renal (re)absorption of amino acids is highlighted by the fact that mutations in either the rBAT or b degrees (,+)AT subunit result in cystinuria whereas a defect in the y(+)-LAT1 light chain causes lysinuric protein intolerance. Here we investigated the localization of these transporters in intestine since both diseases are also characterized by altered intestinal amino acid absorption. Real time PCR showed organ-specific expression patterns for all transporter subunit mRNAs along the intestine and Western blotting confirmed these findings on the protein level. Immunohistochemistry demonstrated basolateral coexpression of 4F2hc, LAT2 and y(+)-LAT1 in stomach and small intestine, whereas rBAT and b degrees (,+)AT were found colocalizing on the apical side of small intestine epithelium. In stomach, 4F2hc and LAT2 were localized in H(+)/K(+)-ATPase-expressing parietal cells. The abundant expression of several members of the heterodimeric transporter family along the murine small intestine suggests their involvement in amino acids absorption. Furthermore, strong expression of rBAT, b degrees (,+)AT and y(+)-LAT1 in the small intestine explains the reduced intestinal absorption of some amino acid in patients with cystinuria or lysinuric protein intolerance.

Lysinuric protein intolerance: mechanisms of pathophysiology.

Heteromeric amino acid transporters (HATs) are composed of two subunits, a polytopic membrane protein (the light subunit) and a disulfide-linked type II membrane glycoprotein (the heavy subunit). HATs represent several of the classic mammalian amino acid transport systems (e.g., L isoforms, y(+)L isoforms, asc, xc-, and b(0,+)). The light subunits confer the amino acid transport specificity to the HAT. Two transporters of this family are relevant for inherited aminoacidurias. Mutations in any of the two genes coding for the subunits of system b(0,+) (rBAT and b(0,+)AT) lead to cystinuria (MIM 220100). Transport defects in a system y(+)L isoform, composed of 4F2hc and y(+)LAT-1, result in lysinuric protein intolerance (LPI) (MIM 222700). In this case, only mutations in the light subunit y(+)LAT-1, but not in the heavy chain 4F2hc, cause the disease. LPI, in addition to affecting intestinal and renal reabsorption of amino acids, is a multisystemic disease affecting the urea cycle and presents also with symptoms related to the immune system. The pathogenesis of these alterations is less well, or not understood at all.